Temporary protection or blocking of chemically reactive functions in biological compounds is an important tool in the field of biological chemistry. To this end, researchers have developed a number of protecting groups. The vast majority of the known protecting groups, however, are acid or base labile and while there are also protecting groups that are labile under neutral conditions, most of these protecting groups are also somewhat acid and base labile. Greene, T W, “Protective Groups in Organic Synthesis”, publishers Wiley-Interscience (1981). Furthermore, many protecting groups suffer additional synthesis, side-reaction, and/or solubility problems. For example, only a few protecting groups applied as a part of a linking system between the solid phase and the oligonucleotide can withstand all the rigors of oligonucleotide synthesis and deprotection thereby facilitating the final purification of oligonucleotides free of truncated or depurinated fragments. See “Solid Phase Synthesis,” Kwaitkowski et al., PCT International Publication WO 98/08857 (1996). Selective post-synthetic derivatization of oligonucleotides also requires selectively cleavable protecting groups. See, e.g., Kahl & Greenberg, “Introducing Structural Diversity in Oligonucleotides via Photolabile, Convertible C5-substituted Nucleotides,” J. Am. Chem. Soc., 121(4), 597-604 (1999).
Protecting groups also should be removable. Ideally, the protecting group is removable under mild conditions, for example, without disturbing interactions between biomolecules. These types of protecting groups may be useful for deprotecting oligonucleotides without disturbing interactions between oligo/polynucleotide strands. For example, International Publication WO 96/23807 entitled “Novel Chain Terminators, The Use Thereof for Nucleic Acid Sequencing and Synthesis and a Method of their Preparation” discloses methods that use nucleotides that are reversibly blocked at the 3′ hydroxyl group. These reversibly blocked nucleotides can be used in sequencing methods where, unlike the well-known Sanger sequencing method that utilizes terminating dideoxynucleotides, the temporarily 3′-OH-protected intermediates can be converted into nucleotides having a free 3′-OH that may be further extended.
One such sequencing method that uses reversibly blocked nucleotides is known as Sequencing by Synthesis (SBS). SBS determines the DNA sequence by incorporating nucleotides and detecting the sequence one base at a time. To effectively sequence long stretches of a nucleic acid using SBS, it is advantageous to be able to perform multiple iterations of the single nucleotide incorporation. Accordingly, SBS-based methods require 3′-OH protecting groups that are removable under conditions that do not distrupt the primer and target DNA interactions. As such, there exists a need for nucleotide triphosphates that are reversibly blocked at the 3′ position and which are also effective substrates for DNA polymerases.